Development of an orally relevant biofilm disinfection model

Abstract

This thesis describes the development and use of a novel microtitre plate biofilm system for testing the antimicrobial activity of test materials. The developed model is capable of high-throughput screening and furthermore the system has been shown to be stable and reproducible. The search for new antimicrobial agents for improved plaque control requires appropriate screening models. Key criteria for these models include; predictive of clinical data, orally relevant organisms (mixed species, bacteria present in biofilms), short contact time, rapid, reproducible and high throughput. The most widely used biofilm system for evaluating oral antimicrobials are the Constant Depth Film Fermentor (CDFF) and the Minimum Biofilm Eradication Concentration (MBEC) model systems. Each system has advantages for specific investingations; however neither and no other single system fulfils all of the criteria listed above. The CDFF is an orally relevant model that mimics biofilm development under constant salivary flow; typically the inoculum is derived from human dental plaque. Microbial analyses of in vitro growth show populations that are representative of in vivo plaque. However, the system is prone to contamination, is labour intensive and has limited capacity for testing multiple agents. The MBEC model investigates the antibacterial susceptibility of attached bacteria to the 96-pegs on the lid of a microtitre plate based system. Unfortunately, this model was not originally designed for use with oral bacteria; therefore, concerns exist for the use of the MBEC with oral bacteria including the development of oral biofilms on non-orally relevant surfaces such as polystyrene, as found in the MBEC pegs. The aim of this project was to develop a microtitre plate based biofilm assay that could assess the effects of antimicrobials against orally relevant biofilms grown on a relevant surface and compare it to recognised standard model systems. Biofilms derived from a defined inoculum were grown on hydroxyapatite-coated microtitre plate wells. Biofilm characteristics were assessed and were shown to be reproducible and allow for high-throughput screening. Antimicrobial testing showed a dose response and known actives were able to be 'ranked' in the same order as seen in clinical trials. This research has culminated in the development of a simple, high-throughput, reproducible 'off-the-shelf' method for the rapid screening of antimicrobial compounds against an orally relevant biofilm.